The overall objective of the proposed research is to understand the mechanism of inheritance oil mitochondrial DMA (mtDNA) using the budding yeast, Saccharomyces cerevisiae, as a model organism. Our studies will combine genetic, biochemical, molecular and cell biological approaches to characterize the various components that contribute to the faithful transmission of mtDNA during vegetative growth. To these ends, we propose the following specific aims: 1) to probe the structure and organization of mtDNA nucleoids. The goal is to understand how the nucleoid proteins are associated with mtDNA, and how they contribute to mtDNA organization and inheritance; 2) to elucidate components of the mtDNA segregation apparatus. We will use a variety of biochemical and cell biological methods to examine how mtDNA nucleoids and certain mutant forms of the nucleoid protein, Ilv5p, which behaves according to all criteria tested like mtDNA nucleoids, interact with the inner mitochondrial .membrane and with candidate proteins that are likely to be components of the mtDNA segregation apparatus; 3) to elucidate the role of the RTG-retrograde response pathway in mtDNA stability and inheritance. We have found that this pathway is intimately involved in mtDNA inheritance through its control of 'metabolic' nucleoid proteins. We will evaluate how the retrograde pathway controls the stability and inheritance of mtDNA, and circumvents the requirement for the major mtDNA packaging protein, Abf2p; 4) to establish the rules of mtDNA nucleoid division and the roles of Hsp60, active ori sequences and transcription of mtDNA in nucleoid division. These objectives follow from our discovery that nucleoid division is a regulated process. We propose to develop both asynchronous and synchronous nucleoid division systems. These studies will be coupled with the analysis of cis-acting elements that distinguish active from inactive ori elements. Altogether, these studies have direct implications for understanding mitochondrial diseases in humans.